Topographical study of intranuclear distribution and compartementalization of viral and cellular proteins

Sammanfattning: The nuclei of eukaryotic cells contain a number of specialized domains involved in DNA replication, transcription, pre-mRNA splicing and ribosome assembly. In addition to these structures, nuclear bodies of unknown function have been described. In the present study, we have used a topographical approach, a combination of immuno-fluorescence and fluorescence digital imaging microscopy (FDIM), to analyze nuclear bodies in cells transformed by Epstein-Barr virus (EBV) and simian virus 40 (SV40). EBV-transformed Iymphoblastoid cell lines (LCLs) express six EBV-encoded nuclear antigens EBNA-1 to -6, the functions of which are not completely known. Detailed analysis of the nuclear patterns of EBNA-1, -2, -3 and -5 has revealed that they differ in their intranuclear distribution. EBNA-5 forms a limited number of large, globular foci, a pattern unique among the EBNAs. The subsequent analysis of the EBNA-5 foci in relation to other nuclear constituents, shows that a subfraction of the retinoblastoma protein (Rb) is concentrated in the EBNA-5 foci in the EBV-transformed LCL IB4. The heat shock protein hsp70 is also present in these foci. Double immunostaining of EBNA-5 and the promyelocytic leukemia associated proteins (PML) in IB4 cells demonstrates that the EBNA-5 foci are surrounded by a PML containing shell (PML body). Moreover, in EBV- infected B Iymphocytes, EBNA-5 accumulates in pre-existing PML bodies. Upon metabolic stress, EBNA-5 is dissociated from PML bodies and translocates to nucleoli. Based on these data, we suggest that in EBV-mediated transformation, EBNA-5 may be involved in the deregulation of cell growth control through: (1) sequestering Rb, and/or (2) targeting PML bodies. We have investigated the possible connection between the PML bodies and another viral transforming protein, the SV40-encoded large T antigen (SV40T), in a conditionally SV40T-immortalized human cell line, IDH4. Nearly all PML bodies are surrounded by 1- 3 SV40T positive foci, suggesting that SV40T may also target PML bodies but in a different manner from EBNA-5. The tumor suppressor protein p53 is also present in the SV40T foci. By modulating the SV40T level in IDH4 cells, we have examined the dynamics of SV40T and p53 compartmentalization. Our observations suggest that the level of p53 is co-regulated with the level of SV40T in a dose-dependent fashion and that SV40T and p53 show a high degree of colocalization. A change in the PML distribution has been observed in IDH4 cells in which the expression of SV40T is suppressed and a senescent phenotype is induced. The majority of these cells have large doughnut- and fibre-like PML structures, as compared to a small fraction of proliferating IDH4 cells. The PML patterns in young, senescent and serum-starved young human fibroblasts suggest that the change in PML distribution is associated with senescence, although it may in part be related to growth arrest. Detailed analysis of the doughnut-like PML structures using 3-D FDIM has revealed that they are actually cylindrical or egg shaped structures in which PML is concentrated in an outer shell. Taken together, the results of the present study show that two different transforming proteins EBNA-5 and SV40T are concentrated in foci that also contain tumor suppressor proteins, Rb and p53 respectively. This phenomenon may reflect complexing and accumulation of these proteins in foci within or close to PML bodies. ISBN 91 -628-2181 -4